Neuritin accelerates Schwann cell dedifferentiation via PI3K/Akt/mTOR signalling pathway during Wallerian degeneration

Abstract Neuritin, also known as candidate plasticity gene 15 (CPG15), was first identified as one of the activity‐dependent gene products in the brain. Previous studies have been reported that Neuritin induces neuritogenesis, neurite arborization, neurite outgrowth and synapse formation, which are involved in the development and functions of the central nervous system. However, the role of Neuritin in peripheral nerve injury is still unknown. Given the importance and necessity of Schwann cell dedifferentiation response to peripheral nerve injury, we aim to investigate the molecular mechanism of Neuritin steering Schwann cell dedifferentiation during Wallerian degeneration (WD) in injured peripheral nerve. Herein, using the explants of sciatic nerve, an ex vivo model of nerve degeneration, we provided evidences indicating that Neuritin vividly accelerates Schwann cell dedifferentiation. Moreover, we found that Neuritin promotes Schwann cell demyelination as well as axonal degeneration, phagocytosis, secretion capacity. In summary, we first described Neuritin acts as a positive regulator for Schwann cell dedifferentiation and WD after peripheral nerve injury.

dedifferentiation and demyelination, while the dedifferentiated Schwann cells plays a vital role in the clearance of the debris of axons and myelin collaborating with macrophages.Other than that, secretion capacity of dedifferentiated Schwann cells is also crucial for subsequent nerve regeneration.In the present study, we first identified the role of Neuritin in Schwann cell dedifferentiation, demyelination, phagocytosis, secretion capacity during WD after peripheral nerve injury.

| Experimental animals and ethics statement
Specific pathogen-free Sprague-Dawley male adult rats (aged 8 weeks) weighing 200-250 g and neonatal rats (postnatal Day 3)   were provided by the Animal Center of Southern Medical University, China (licence No. SCXK (Yue) 2016-0041).The rats were housed in an animal room maintained at 21°C and 55% relative humidity with a 12-h light/dark cycle and given free access to water and food.All procedures, including surgery and tissue collection, were carried out with the approval of the Southern Medical University Animal Care and Use Committee (approval No. SMU-L2015081, approval date 15 October 2015) in accordance with the guidelines for the ethical treatments of animals.All efforts were made to minimize the number of animals used and their suffering.

| Sciatic nerve explant culturing and treatment
Sciatic nerve explant culturing, which is an in vitro model of WD, was performed as described previously. 8,9Briefly, rats were anaesthetised by intraperitoneal injection of 12 mg/mL tribromoethanol (180 mg/ kg; Sigma-Aldrich, St. Louis, MO, USA) and decapitated by guillotine.
According to the previous reports, 200 ng/mL recombinant Neuritin (PeproTech, Rocky Hill, NJ, USA) was added to the culture medium, and the nerve explants were collected after culturing for 5 or 8 days post injury (dpi) for subsequent analysis. 10,11A vehicle-only control group (Con group, medium without neuritin) was included.
The relative protein expression levels were normalized to GAPDH.

| Statistical analysis
All data are presented as mean ± standard error of the mean (SEM).
All statistical analyses were performed with GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA) using one-way analysis of variance followed by Bonferroni's multiple test.A p < 0.05 was considered statistically significant.

| Temporal and spatial expression of Neuritin in ex vivo sciatic nerve explants
The sciatic nerve explants were cultured in vitro for 5 or 8 days (Figure 1A).

| Effects of Neuritin on Schwann cells demyelination and fragmentation
First, the sciatic nerve explants cultured for 5 days were teased into single fibres facilitate visualization of the myelin sheath by phase contrast microscopy (Figure 2A).As myelin ovoids was easily identified and quantified, we found that the number of them present in every 200μm length of teased fibre (the ovoid index) was highest in the Neuritin group than that in the control group (Figure 2B).ORO staining is used to label lipid droplets in adipocytes. 16,17Here, we found that the mean intensity of ORO (an index of degenerated myelin) in the Neuritin group was signifcantly higher than that in the control group (Figure 2C-F).

| Effects of Neuritin on axon and myelin degeneration
Next, we investigated the role of Neurtin in axonal and myelin degeneration in the explant model. 18Immunofuorescence staining on the cross and longitudinal sections showed that the NF staining axons and MBP staining myelin were signifcantly decreased in Neuritin group compared with the control group (Figure 3A-E).Similar trends were observed when NF and MBP levels were assessed by western blot (Figure 3F,G).

| Effects of Neuritin on the Schwann cells capacities of phagocytosis and neurotrophins expression
Recent studies indicated that Schwann cells response to debris clearance, namely phagocytosis.Current findings hinted that both phagocytosis and secretion function could be enhanced by Neuritin in Schwann cells.Both cross and longitudinal sections of the nerve explants showed that the ratio of ORO/S100 double positive Schwann cells and the number of ORO+ myelin debris ingested by each S100+ Schwann cell were significantly higher in the Neuritin group than those of the control group (Figure 4A-E).
Subsequently, western blotting was used to assess the main neurotrophic factors secreted by Schwann cells, such as GDNF, NGF and BDNF.Current results illustrated that the protein levels of the three neurotrophins were all significantly higher in the Neuritin group compared with the control group (Figure 4F-I) which implies that Neuritin promote neurotrophins expression during WD and may play an vital role in the following nerve regeneration.

| Effects of Neuritin on Schwann cell dedifferentiation
As described previously, Schwann cell dedifferentiation and transformation into repair phenotype after nerve injury is a prerequisite for their capabilities of debris clearance and neurotrophins secretion. 19,20Herein, c-Jun (a marker of immature Schwann cells) and MAG (a marker of mature Schwann cells) antibodies were used to identify the role of Neuritin in Schwann cell dedifferentiation via immunostaining and western blotting.Interestingly, the c-Junpositive fluorescent intensity and the number of c-Jun-positive cells in the Neuritin group were remarkably higher than those in the control group.In contrast, the number of MAG-positive cells was significantly lower in the Neuritin group than in the control group (Figure 5A-C).Similarly, the c-Jun expression was drastically upregulated in the Neuritin group, while MAG was obviously decreased (Figure 5D,E).These data vividly indicated that Neuritin facilitates Schwann cell dedifferentiation in the injured nerve.

| Neuritin promotes Wallerian degeneration through PI3K/Akt/mTOR Pathway
Neuritin is well known for its importance in neurons by regulating the neuronal excitability through PI3K/Akt/mTOR pathway. 21,22owever, whether Neuritin accelerates the process of WD through PI3K/Akt/mTOR pathway is unknown yet.To test the hypothesis, we compared the expression levels of PI3K/Akt/mTOR by western blotting.As expected, PI3K/Akt/mTOR were dramatically increased in Neuritin group compared to the control group (Figure 6A-D).
Next, mTOR inhibitor rapamycin and PI3K inhibitor LY294002 were individually used to assess their effects on the Schwann cells demyelination.As a result, we found that both rapamycin and LY294002 impeded the neuritin induced accelerated demyelination (Figure 6E-G).Above data indicate that Neuritin can up-regulate the expression of PI3K/Akt/mTOR, which results in promoting the process of WD.

| DISCUSS ION
WD, an essential reaction response to nerve injury, is very important for subsequent nerve repair and regeneration. 6,23Until now, many critical factors (e.g.Schwann cells demyelination, dedifferentiation and phagocytosis) for WD have been identifed. 24,25Schwann cells undergoes a particular plasticity response and is crucial for the successful regeneration of the peripheral nervous systerm.
Neuritin is upregulated in hippocampal and cortical neurons in vivo and in vitro by the neurotrophin brain-derived neurotrophic factor 20 and induces neurite outgrowth.Although the role of Neuritin as key regulator for neuron regeneration after PNI is well investigated, whether Neuritin also plays a role in Schwann cells during WD remains unclear.In the present study, we found that Neuritin expression was upregulated at 5 or 8 dpi after sciatic nerve injury.Neuritin is predominately located in the perinuclear cytoplasm, Schmidt-Lanterman incisures, inner mesaxons, outer mesaxons in the normal peripheral nerve fibres.From 5 to 8 dpi, Neuritin is mainly diffused around the myelin ovoids.Since previous studies demonstrated that the first 7 days post nerve injury is a critical period for WD, 26  NF, MAG, and MBP expression which are selected to reflect the levels of axons and myelin, respectively, since axonal regeneration and remyelination might occur before WD is completed. 27Moreover, circulating macrophages cannot be recruited into the nerve explants to accelerate WD, so the differences among each group are mainly attributed to the effects of Neuritin on Schwann cells.
In this model, the collected results easily drive us to believe that Neuritin plays a positive role in WD since Neuritin significantly increased the proportion of degenerated myelin and decreased expression levels of NF and MBP, all of which meant the process of WD was accelerated.Meanwhile, immunostaining and western blotting with antibodies to MAG (for differentiated Schwann cells) and c-Jun (for dedifferentiated Schwann cells) indicated Neuritin enhanced the Schwann cell dedifferentiation.WD occurs at the early stage after nerve injury, which can create a favourable microenvironment for the following nerve regeneration.2][33] Further, recently study founded that Neuritin suppresses oesophageal cancer growth and up-regulates Kv4.2-mediated transient outward K + current through the PI3K/Akt/mTOR pathway in rat cerebellar granule neurons. 11In order to explore whether Neuritin plays a role in the Schwann Temporal and spatial expression of Neuritin in sciatic nerve explants.(A) Establishment of ex vivo nerve explants, the sciatic nerves from 8 W rats were equant cut into segments with 3 mm length and cultured in Schwann cells medium.(B) Western blot of proteins from injured sciatic nerve at 5 and 8 dpi probed with antibodies against Neuritin.(C) Quantifcation of Neuritin protein levels in the blots.(D) Representative images showing that total Neuritin is upregulated increasingly during 5-8 dpi.And Neuritin is potently enriched in the perinuclear cytoplasm and diffused in inner mesaxon, outer mesaxon and Schmidt-Lanterman incisure (n = 3, *p < 0.05).
Protein extracts from the collected nerve segments, as well as the in vitro cultured nerve explants, were prepared by routine procedures, then separated on 10% dodecyl sulfate sodium saltpolyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes (BioRad, Hercules, CA, USA).After blocking with 5% bovine serum albumin (GBCBIO, Guangzhou, F I G U R E 2 Neuritin promotes myelin degeneration in nerve explants.(A and B) Teased fibres from sciatic nerve explants treated with Neuritin showing more myelin ovoids compared with those of Con group.(C-F) ORO staining showed the myelin sheath degeneration (shrunken myelin with high fluorescence intensity) appeared in nerve explants.And statistical analysis illustrating that the levels of myelin degeneration are signifcantly higher in Neuritin group than those of the Con group.(n = 4, *p < 0.05).

| 5 of 9 LIU
Aiming to understand the functional role of Neuritin during WD in ex vivo nerve explants, we firstly assessed the expression F I G U R E 3 Neuritin promotes axon and myelin degeneration in nerve explants.(A) Immunohistochemistry images showing fragmented axons and myelin sheaths in transverse and longitudinal sections of sciatic nerve explants treated with Neuritin at 5 dpi.(B and C) Mean fluorescence intensity and ratio of double staining of axons and myelin fragments in transverse sections showed that Neuritin could more dramaticly accelerate degeneration compared with Con group.(D and E) Quantifcation of the length distributions of axons and myelin fragments showing both the length of axonal fragments and myelin fragments are signifcantly shorter in the Neuritin group than those of the Con group.(F and G) Western blots and statistical analysis illustrating that the levels of MBP and NF protein are signifcantly lower in Neuritin group than those of the Con group.(n= 4, *p < 0.05).et al. of total Neuritin by western blotting.Results showed that a significant increase of protein levels in the sciatic nerve explants from 5 days post injury (dpi) to 8 dpi (Figure 1B,C).To localize the expression of Neuritin, we performed immunofluorescent staining on teased sciatic nerve fibres.Briefly, after cultured for 5 or 8 days, nerve fibres were stained with fluorescein phalloidin and Neuritin.Results showed that Neuritin was potently enriched in perinuclear cytoplasm, Schmidt-Lanterman incisures, inner mesaxons, outer mesaxons at 0 dpi.However, the immunoreactivities of Neuritin were mainly diffused mostly around the myelin ovoids, which are the typical structure of a fragmented myelin sheath (Figure 1C,D).These findings indicate that Neuritn is meaningful for WD after peripheral nerve injury.F I G U R E 4 Neuritin facilitates Schwann cells capabilities of phagocytosis and secretion.(A-E) S100 and Lum double staining and quantitative analysis after the Schwann cells cultured with lum at 5 dpi in vitro illustrated that phagocytosis of Schwann cells were significantly enhanced in the Neuritin group compared with the Con group.(F-I) Western blots and statistical analysis illustrated that the levels of GDNF, NGF, BDNF protein are signifcantly higher in Neuritin group than those of the Con group.(n = 4, *p < 0.05).

F I G U R E 5 | 7 of 9 LIU
Neuritin facilitates Schwann cell dedifferentiation.(A-C) Immunofluorescence and quantification showed that the number of MAG-positive cells was significantly lower in the Neuritin group than in Con group.In contrast, the number of c-Junpositive cells in the Neuritin group were remarkably higher than those in Con group.(D and E) Western blots and quantification showed the protein levels of MAG are signifcantly lower in Neuritin group than those of the Con group, while c-Jun was obviously increased in the Neuritin group.(n= 4, *p < 0.05).et al.
therefore, our data suggest the high expression of Neuritin in this critical period might be meaningful for Schwann cell demyelination and the following WD.In order to figure out this issue, we used a simplified ex vivo WD model of sciatic nerve explants to test the effects of Neuritin on the WD as well as the Schwann cell dedifferentiation.In this model, not any nerve regeneration will occur to interrupt the evaluation of the F I G U R E 6 Neuritin activates the PI3K/Akt/mTOR signalling pathway after peripheral nerve injury.(A) Western blots assay was performed in the distal trunk of the injured sciatic nerves and indicated the expression level of PI3K/Akt/mTOR.(B-D) Statistical analysis illustrated that the protein levels of PI3K, p-Akt/AKT, p-mTOR/mTOR are signifcantly higher in Neuritin group than those of the Con group.(E-G) ORO staining showed that Neuritin promotes SCs demyelination but inhibited by PI3K inhibitor LY294002 or mTOR inhibitor rapamycin.(n= 4, *p < 0.05).
cell demyelination during WD is caused by regulating PI3K/Akt/mTOR pathway, we set up the ex vivo nerve explants model and found that an up-regulation of PI3K, p-Akt, p-mTOR was revealed by western blotting in Neuritin group.Finally, we used mTOR inhibitor rapamycin and PI3K inhibitor LY294002 were individually used to assess their effects on the Schwann cells demyelination, by which the effect of Neuritin of the Schwann cell demyeliation and WD was efficiently reversed.In conclusion, present data indicate that Neuritin plays a positive role in WD by up-regulating the PI3K/Akt/mTOR signalling pathway.Thus, our results provide a new insight into Neuritin regulation of peripheral nerve degeneration and suggest a potential therapeutic target for recovery of peripheral nerve injury.